This relates to the mammalian insulin receptor. In particular it relates to the synthesis of the insulin receptor by recombinant cells and to the manufacture and use of certain novel products enabled by the identification and cloning of DNA encoding the insulin receptor.
The rapid matabolic effects of insulin as well as its long term growth promoting actions are initiated by the interaction of the hormone with specific, high affinity cell surface receptors.sup.1-3. The indulin receptor (apparent M.sub.r 350,000-400,000) is an integral membrane glycoprotein composed of two .alpha. subunits (apparent M.sub.r =120,000-130,000) and two .beta. subunits (apparent M.sub.r =90,000), which are linked by disulfide bonds .sup.4-8. Photoaffinity labelling of the receptor.sup.9,10 as well as affinity cross linking.sup.7,11 have shown that the .alpha. submit is predominantly labelled by radioactive insulin derivatives. In intact cells, insulin stimulates the phosphorylation of the .beta. subunit on serine and tyrosine residues, a phenomenon first observed in rat hepatoma cells and IM9 lymphoblasts.sup.12,13, and then extended to a variety of cell types.sup.14-16. In vitro, insulin-dependent tyrosine kinase activity.sup.14-22 copurifies with the insulin-binding activity to homogeneity.sup.19,20,23. The protein kinase activity of the insulin receptor catalyzes both phosphorylation of the .beta. subunit as well as exogenous peptides and proteins.sup.14,21,24-26. Studies with model peptide substrates indicate that the specificity of the insulin-dependent protein kinase is similar to that of the epidermal growth factor (EGF) receptor kinase and the src family of tyrosine specific protein kinases.sup.24,25. In addition to being the principal substrate for autophosphorylation, the .beta. subunit bears an ATP binding site.sup.21,22.
The .alpha. and .beta. subunits of the heterotetrameric insulin receptor derive from a single, glycosylated, polypeptide precursor of approximately 190,000 daltons.sup.29-31. Disulfide bridges linking the .alpha. and .beta. subunit regions probably begin to form as the protein folds. After being transported from the endoplasmic reticulum to the Golgi apparatus, the precursor is further glycosylated and is then cleaved, followed by its transport to the plasma membrane.sup.27. Although similar in purported structure to the IGF-1 receptor.sup.28, the insulin receptor differs from the EGF receptor, which functions in the plasma membrane as a single polypeptide chain.
The amino acid sequence of the human EGF receptor was recently reported .sup.32,33. However, although a need has existed for identifying the DNA encoding the insulin receptor and for expressing the DNA in recombinant culture, and notwithstanding reports of purified human placental insulin receptor in 1981 (7), no sequence information for this complex protein as yet has been disclosed in the literature